Identifying mechanisms of contact-mediated cell polarization - Resubmission

项目总结 从细胞的不对称分裂到组织的形成，极性的建立对于发育是必不可少的。 PAR极性蛋白的径向极化存在于早期哺乳动物胚胎中，是分离所必需的 胚胎血统和胚胎外血统。早期哺乳动物胚胎和上皮细胞使用细胞接触作为 建立极性的线索，但细胞接触线索和皮质PAR极性之间的分子联系 使细胞极化的蛋白质仍然知之甚少。线虫的早期胚胎极化时，RhoGAP， PAC-1/ARHGAP21被招募到细胞接触(充当对称破坏信号)并使CDC-42失活， 一种Rho-GTP酶，导致接触部位的PAR蛋白丢失。E-钙粘附素，一种高亲和性黏附 在许多不同的细胞类型中，接触介导的极性都需要蛋白质。在线虫中，E-钙粘蛋白扮演着一种 通过帮助招募PAC-1到接触地点，在建立极性方面发挥指导作用。E-钙粘附素作用于 钙粘附素-连环蛋白复合体(CCC)的组成部分，用于驱动极化，并与 定位PAC-1的未知钙粘附素非依赖性途径。参与极性建立的蛋白质 线虫是高度保守的，因此了解它们在这个遗传易驯化系统中的作用将提供 对哺乳动物胚胎和上皮细胞接触介导极化的洞察。这件事的首要目标是 建议确定将细胞接触与细胞内的根尖基极性联系起来的机制。至 实现这一点，我将确定E-钙粘蛋白和PAC-1之间的生化联系，从而将其招募到细胞中 联系人(目标1)。我推测，保守的极性蛋白afadin(AFD-1)通过以下方式帮助PAC-1定位 作为α-连环蛋白/HMP-1(E-钙粘蛋白下游)和PAC-1之间的连接物。我要测试一下这个 假说使用基因操作的CCC和免疫沉淀来确定蛋白质的相互作用。这就做 还可以使用质谱仪鉴定与AFD-1相互作用的蛋白质，以推动PAC-1的定位和潜在功能。 我将确定也定位PAC-1的E-钙粘素非依赖性途径(目标2)。我假设另一个人 极性蛋白直接(蛋白质-蛋白质相互作用)或间接(通过磷酸化)招募PAC-1 或膜曲率的变化)。我将检查通过酵母二号鉴定的候选蛋白质 与PAC-1的PH结构域相互作用的杂交体(已知的该区域在没有E- 在缺乏E-钙粘蛋白的情况下，它们能够定位PAC-1。我还会用质谱仪来鉴定 与PAC-1相互作用的蛋白质是否有E-钙粘附素。这一点，连同磷酸化分析，将允许 我们需要了解PAC-1是如何在没有E-钙粘附素的帮助下定位的。一种无偏EMS诱变筛选方法 E-钙粘蛋白缺失的背景将有助于确定在缺乏E-钙粘蛋白的情况下PAC-1如何定位。这些 这些发现将为细胞接触线索如何使胚胎和上皮细胞两极分化提供基本的见解。

PROJECT SUMMARY Polarity establishment is essential for development, from asymmetric cell division to the formation of tissues. Radial polarization of PAR polarity proteins occurs in early mammalian embryos and is required for separation of embryonic and extra-embryonic lineages. Early mammalian embryos and epithelial cells use cell contacts as cues for establishing polarity, but the molecular links between cell contact cues and the cortical PAR polarity proteins that polarize cells remain poorly understood. The C. elegans early embryo polarizes when the RhoGAP, PAC-1/ARHGAP21, is recruited to cell contacts (acting as the symmetry breaking cue) and inactivates CDC-42, a Rho-GTPase, resulting in the loss of PAR proteins from contact sites. E-cadherin, a homophilic adhesion protein, is required for contact-mediated polarity in many different cell types. In C. elegans, E-cadherin plays an instructive role in establishing polarity by helping to recruit PAC-1 to contact sites. E-cadherin acts with components of the cadherin-catenin complex (CCC) to drive polarization, and functions redundantly with an unidentified cadherin-independent pathway to localize PAC-1. The proteins involved in polarity establishment in C. elegans are highly conserved, so understanding their role in this genetically tractable system will provide insights into contact-mediated polarization in mammalian embryos and epithelia. The overarching goal of this proposal is to identify mechanisms that link cell contact with apicobasal polarity within the cell. To achieve this, I will identify the biochemical connections between E-cadherin and PAC-1 that recruit it to cell contacts (Aim 1). I hypothesize that the conserved polarity protein, afadin (AFD-1), aids in PAC-1 localization by functioning as a linker between a-catenin/HMP-1 (downstream of E-cadherin) and PAC-1. I will test this hypothesis using genetic manipulation of the CCC and immunoprecipitation to identify protein interactions. I will also use mass spec to identify proteins interacting with AFD-1 to drive PAC-1 localization and potentially function. I will identify the E-cadherin-independent pathway that also localizes PAC-1 (Aim 2). I hypothesize that another polarity protein recruits PAC-1, either directly (protein-protein interaction) or indirectly (through phosphorylation or changes in membrane curvature). I will examine candidate proteins that were identified through yeast two hybrid to interact with the PH domain of PAC-1 (the region known to be important for localization without E- cadherin) for their ability to localize PAC-1 in the absence of E-cadherin. I will also use mass spec to identify the proteins interacting with PAC-1 with and without E-cadherin. This, along with phosphorylation analysis, will allow us to understand how PAC-1 localizes without the aid of E-cadherin. An unbiased EMS mutagenesis screen in an E-cadherin null background will aid in determining how PAC-1 localizes in the absence of E-cadherin. These findings will provide fundamental insights into how cell contact cues can polarize embryos and epithelial cells.